Antioxidant Measurement and Applications - American Chemical Society

Chapter 16

Downloaded by UCSF LIB CKM RSCS MGMT on November 19, 2014 | http://pubs.acs.org Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch016

Peanut Skin Phenolics: Extraction, Identification, Antioxidant Activity, and Potential Applications Jianmei Yu, Mohamed Ahmedna, and Ipek Goktepe Food and Nutrition Sciences, North Carolina Agricultural and Technical State University, Greensboro, NC 27411

Extraction and determination of total phenolics (TP) in peanut skins obtained from various processing methods was conducted. Total antioxidant activity (TAA) of peanut skin extracts was determined in order to identify and quantify different classes of phenolics in peanut skin. Peanut skins were removed by direct peeling, blanching, and roasting. TP and T A A of peanut skin extracts were determined using Folin-Ciocalteu and free radical scavenging methods, respectively. The composition of peanut skin extracts was determined by L C - M S . Peanut skin processing significantly affected total extractable phenolics and their composition. Roasting had limited effects on TP while blanching caused 89% loss of TP. TP in directly peeled, roasted, and blanched peanut skins were 130, 124, and 14.4 mg/g, respectively. T A A of all purified peanut skin extracts were higher than those of Trolox and Vitamin C at equivalent concentrations. L C - M S analysis of purified peanut skin extracts revealed the presence of catechins, A-type and B-type procyanidin dimers, trimers and tetramers. Total catechins, procyanidin dimers, trimers and tetramers in directly peeled peanut skin were 16.1, 111.3, 221.3 and 316.1 mg/100g, respectively, versus 8.8, 143.5, 157.5 and 203.9 mg/100 g, respectively, in roasted skin.

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© 2007 American Chemical Society In Antioxidant Measurement and Applications; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


227 Peanut is an important crop grown in the U.S. and worldwide. In 2005, peanut production in the United States was about 4.49 billion pounds (7). Georgia, Texas, Alabama, and North Carolina are the largest peanut producers in the United States. The edible part of peanuts consists of the kernel and protective skin. The peanut kernels are used to make peanut butter, roasted snack peanuts, peanut confections and peanut oil. The skin has a pink-red color with astringent taste, and is typically removed before peanut consumption. Peanut skin represents 3-7% of peanut kernel weight depending on the variety and size of the peanuts. Peanut skin, as a low cost peanut processing industry, contains 12% protein, 16% fat, 72% of carbohydrate, rich in polyphenols (2), and is mainly used as animal feed (3,4). The high polyphenol content of peanut skin may provide a potent source of antioxidants for use in foods and dietary supplements. Despite it high polyphenol content, limited studies have been carried out to evaluate the content and activity of peanut skin phenolics. For instance, six A type procyanidin isomers were identified in peanut skin by Lou et al. (5) using C N M R . These six compounds were found to inhibit the activity of hyaluronidase, an enzyme that responsible for the release of histamine which causes inflammation. In addition, resveratrol, a phytochemical found in grape seed and wine, was also found in peanut skin in much higher concentration than in peanut kernels (6). Previously published work indicated that the total phenolics of non-defatted peanut skin was about 90-125 mg/g dry skin, including phenolics acids, flavonoids and resveratrol (7). Catechins, B-type procyanidin dimmers, procyanidins trimers, tetramers and oligomers with higher degree of polymerization were also reported in peanut skin (8). Procyanidins are polyphenolic compounds generally found in pine bark (9), grape seeds and skin (70-77), as well as cocoa, cranberries and apples (72). Monomeric procyanidins are polyhydroxyflan-3-ol monomers, and are generally known as catechins. A type procyanidins are formed through a 4—>8 C-C bond and an interflavonoid C13

0 bond (5,13). Procyanidins formed through 4—>8 or 4—>6 C-C bonds of flan-301 monomers are B-type procyanidins. Oligmeric procyanidins (OPCs) including dimmers, trimers and tetramers are also known as proanthocyanidins. These studies indicate that peanut skin could represent an inexpensive source of health promoting phenolics since peanut skin is a low cost by-product of the peanut industry. Quantitative analysis of catechins and procyanidins in peanut skin has not been reported in the literature. Both identification and quantification of catechins and procyanidins in peanut skin are needed to determine their potential as functional ingredients in food, dietary supplement and non-food applications. In addition, the effects of processing methods commonly used to remove skin from peanut kernel on polyphenols retention and their antioxidant activity have not been reported in the literature. This study was conducted to determine the effects of three peanut processing methods on the composition of peanut; to identify/quantify peanut skin procyanidins compounds under each processing

In Antioxidant Measurement and Applications; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

228 and concentration process, and to evaluate the total antioxidant activity and free radical scavenging capacity of all peanut skin extracts.

Materials and Methods


Materials Un-peeled raw peanuts were purchased from a local grocery store (Greensboro, N C ) . Standards including gallic acid (GA), catechin (C), epicatechin (EC), resveratrol, caffeic acid, p-coumaric acid, ferulic acid, ellagic acid and chlorogenic acid, and reagents 2,2'-Azino-bis-(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 2,2-Diphenyl-2-picrylhydrazyl (DPPH) free radical and peroxidase (EC 1.11.1.7) from horseradish were purchased from Sigma Chemical Co. (St. Louis, MO). Procyanidin B2 was purchased from INDOFINE Chemical Company (Hillsborough, NJ). Trolox, hydrogen peroxide (30%), methanol, ethanol, acetonitrile, trifluoroacetic acid and acetic acid were purchased from Fisher Scientific (Atlanta, G A ) . Removal of Peanut Skins Peanuts skins were removed from kernels by three methods: direct peeling (DP), blanching (B) and roasting (R). In the direct peeling method, the skin was directly peeled by hands from raw peanut kernels, then freeze dried. For the blanching method, raw peanuts were boiled in water for 2 minutes, drained and their skin was hand-peeled then freeze dried. The roasting method consisted of heating peanuts at 175°C for 5 min, cooled to room temperature, then rubbing dry skin off. Further drying was not needed for the skin obtained by this method. Dry skins obtained from each method were separately blended into powder and stored at -20 °C in plastic bottles until used. Extraction and Purification of Phenolics Phenolics in peanut skin were extracted using 80% ethanol. Thirty milliliters of solvent were added in a centrifuge tube (50 ml capacity) containing 1.00 g of skin powder. The mixture was homogenized using a Polytron Homogenizer (Polytron PT-MR2100, Kinematica A G , Switzerland) for 2 min then centrifuged at 3000 g for 15 min at room temperature. The supernatant was collected in an amber bottle. The precipitate was subsequently extracted twice by the same procedure described above. The combined supernatant was filtered through a Whatman Anotop syringe filter (0.2 μπι) and stored in a freezer until use. The extraction of each peanut skin was performed in triplicate.


229 The crude alcoholic extracts were evaporated under reduced pressure, and then purified by the method of Hara (14) to remove sugars, proteins and lipids. Phenolic acids and water soluble anthocyanins may have also been removed. The purified extracts were quantitatively re-dissolved in methanol and stored in amber glass vials in a freezer for subsequent analysis. The above extraction and purification process was completed under dim light to minimize light induced degradation/oxidation of phenolics, which are generally light sensitive.


Total Phenolics of Crude and Purified Peanut Skin Extracts Concentrations of total phenolic in crude and purified extracts were determined by Folin-Ciocalteu method (14) using gallic acid as the standard. Results were expressed as mg/mL of gallic acid equivalent (GAE). Free radical scavenging capacity and total antioxidant activity ( T A A ) of peanut skin extracts, total antioxidant activity ( T A A ) of crude and purified peanut skin extracts were determined by two free radical scavenging methods: A B T S * method (16) and DPPH" method (17). Free radical scavenging capacity (FRSC) was calculated as the percentage of color reduction relative to control FRSC% = [ A /A i ] χ 100 in comparison with that of Trolox and ascorbic acid used at the same concentration. T A A was calculated as Trolox equivalent antioxidant activity (TEAC), m M Trolox per m M of total phenolics. +

extract

contro

LC-MS Identification and Quantification The identification of the active compounds in peanut skin extracts was conducted using an Agilent 1100 L C / M S system (Agilent Technologies, Palo Alto, C A ) equipped with HP ChemStation for data collection and analysis. The column used was a Nucleosil RP C (250 mm χ 4.6 mm, and particle size of 5 μπι, Alltec, Deerfield, IL). A binary mobile phase was used to elute injected samples. Mobile phases A and Β were 0.1% formic acid in distilled water and 0.1%) formic acid in 100% acetonitrile, respectively. The flow rate of mobile phase was 0.8 mL/min. The elution gradient was started with 88% of A and 12% of B . Mobile phase Β increased to 15% in 15 minutes, then to 20% in 10 minutes at which it was maintained for 5 min. Afterward, mobile phase Β was increased to 35% in 10 min, maintained at 35% for 5 min, and returned back to 12% in 5 min. The total run time was 50 min. Molecular weights of peaks resolved by L C were determined using Electron Spray Ionization (ESI) Mass Detector working at a negative mode (deprotonation) using both scan mode and selected ion monitoring. The mass range of m/z 5-2000 dalton was scanned at 1 dalton per second. Resolved peaks were detected by Waters 2487 Dual wavelength detector at wavelength 280 nm. The concentrations of catechin and epicatechin were expressed as (+)-catechin equivalents while procyanidin dimmers, trimers and tetramers were expressed as procyanidin B2 equivalent. 1 8


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Results and Discussions Effect of processing on total phenolic content in crude peanut extracts. Direct peeling, blanching and roasting represent three most commonly used methods for peanut skin removal. Table I shows that total phenolics (TPs) were 131, 15 and 124 mg G A E /g dry skin for directly peeled, blanched, and roasted peanut skins, respectively. Total phenolics recovery obtained by the procedure used in this study was higher than that obtained by the procedure of Nepote et al. (118 mg/g dry skin) (18). With the exceptions of blanched peanut skin, the TPs of peanut skin tested in this study compared to that reported by Nepote et al. (150 mg per g defatted dry skin) (2) and significantly higher than the TPs of grape seeds. The latter was reported to be in the range of 5 to 8% of the dry weight of the seeds (19).

Table I. Total phenolics in peanut skins as affected by skin removal methods and sequential extraction Total phenolics (mg G A E I g dry skin)* Skin removing method Direct peeling Peeling after blanching Peeling after roasting

s

I ' extraction

2

103.80 ± 1.80 (79.34%) 8.77 ± 0 . 5 2 (58.22%) 101.47 ± 0 . 8 7 (81.65%)

21.37 ± 2 . 4 5 (16.33%) 4.62 ± 0.36 (30.66%) 17.72 ± 0 . 7 0 (14.26%)

nd

extraction

rd

3 extraction

Total**

5.65 ± 0 . 1 1 (4.32%) 1.68 ± 0 . 1 6 (11.15%) 5.08 ± 0 . 5 3 (4.09%)

130.82 ± 4.16" (100%) 15.07 ± 1.03 (100%) 124.27 ± 2.10" (100%) b

* Total phenolics expressed as mg gallic acid equivalent per g dry skin (GAE/g); value in parentheses represent % TP with respect to cumulative TP in 3 extractions. ** means with same letter superscripts are not significantly different at p

Antioxidant Measurement and Applications - American Chemical Society

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